U.S. patent number 5,003,056 [Application Number 07/288,786] was granted by the patent office on 1991-03-26 for antibiotic nk86-0279, process for production of the same and application of the same.
This patent grant is currently assigned to Nippon Kayaku Kabushiki Kaisha. Invention is credited to Takashi Harada, Kiyonobu Hirose, Takashi Kurokawa, Takaaki Nishikiori, Seiichi Saito, Nobuyoshi Shimada, Takako Tsuchiya, Masanori Yamazaki, Takumi Yamshita.
United States Patent |
5,003,056 |
Nishikiori , et al. |
March 26, 1991 |
Antibiotic NK86-0279, process for production of the same and
application of the same
Abstract
This invention relates to an antibiotic NK86-0279 of the
formula: ##STR1## which exhibits antifungal, antitumor,
vascularizations-inhibitory and insecticidal activities.
Inventors: |
Nishikiori; Takaaki (Tokyo,
JP), Yamazaki; Masanori (Tokyo, JP), Saito;
Seiichi (Kashiwa, JP), Shimada; Nobuyoshi (Tokyo,
JP), Kurokawa; Takashi (Ageo, JP), Hirose;
Kiyonobu (Ageo, JP), Yamshita; Takumi (Tokyo,
JP), Tsuchiya; Takako (Kyoto, JP), Harada;
Takashi (Tokyo, JP) |
Assignee: |
Nippon Kayaku Kabushiki Kaisha
(Tokyo, JP)
|
Family
ID: |
18177105 |
Appl.
No.: |
07/288,786 |
Filed: |
December 22, 1988 |
Foreign Application Priority Data
|
|
|
|
|
Dec 24, 1987 [JP] |
|
|
62-325459 |
|
Current U.S.
Class: |
536/71; 435/76;
435/886; 536/14; 536/18.1; 435/253.5; 514/27; 536/16.8 |
Current CPC
Class: |
C07D
493/20 (20130101); C07H 19/01 (20130101); C12N
1/205 (20210501); C12P 17/181 (20130101); Y10S
435/886 (20130101); C12R 2001/465 (20210501) |
Current International
Class: |
C07H
19/00 (20060101); C07D 493/20 (20060101); C07D
493/00 (20060101); C12P 17/18 (20060101); C07H
19/01 (20060101); C12P 001/06 (); A61K 031/70 ();
A61K 031/71 () |
Field of
Search: |
;536/16.8,18.1,14,7.1
;514/27 ;435/886,76,253.5 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Chem. Abs., vol. 107, 1987, p. 333, Abs. No. 112298n, "Oligomycin
E, a New Antitumor Antibiotic Produced by Streptomyces sp.
MCI-2225", & J. Antibiot. 1987, 40(7), 1053-7 (Abstract). .
Chem. Abs., vol. 96, 1982, p. 292, Abs. No. 65333n, "Trioxacarcins,
Novel Antitumor Antibiotics, I. Producing Organism, Fermentation
and Biological Activities", & J. Antibiot. 1981, 34(12),
1519-24 (Abstract). .
Chem. Abs., vol. 88, 1978, p. 180, Abs. No. 59931z, "Biological
Properties of Culture 6734-21 and Conditions for the Isolation of
the Antiviral Antibiotic Produced by It", & Antibiotiki
(Moscow) 1977, 22(12), 1063-5 (Abstract). .
Chem. Abs., vol. 99, 1983, p. 491, Abs. No. 15683g, & JP-A-58
65 293 (Kyowa Hakko Kogyo Co., Ltd.) 18-04-1983. .
EP-A-O 058 518 (Merck) (Whole document). .
Chem. Abs., vol. 96, 1982, p. 292, Abs. No. 65334p, "Trioxacarcins,
Novel Antitumor Antibiotics. II. Isolation, Physicochemical
Properties and Mode of Action", & J. Antibiol. 1981, 34(12),
1525-30. .
The Journal of Antibiotics, vol. XL, No. 7, pp. 1053-1057..
|
Primary Examiner: Griffin; Ronald W.
Attorney, Agent or Firm: Nields; Henry C.
Claims
We claim:
1. An antibiotic NK86-0279 of the formula: ##STR3##
2. An antifungal agent, an antitumor agent, a vascularization
inhibitor or an insecticide comprising an antibiotic NK86-0279 as
an active ingredient and excipient.
Description
FIELD OF THE INVENTION
This invention relates to a novel antibiotic NK86-0279, a process
for the production of the same and an application of the same.
The compound according to the present invention has antifungal,
antitumor and vascularization-inhibitory and insecticidal effects
Thus it is expected to be available as agrochemicals such as an
insecticide and fungicide or a chemotherapeutic agent against fungi
or malignant tumors or in preventing and/or treating diseases
caused by vascular neoplasia such as nodose rheumatism, diabetic
retinopathy, retinopathy of prematurity, senile macular
degeneration or hypercicatrization during the healing of wound.
BACKGROUND OF THE INVENTION
Known antifugal antibiotics include amphotericin B and nystatin A1
while known antitumor antibiotics include cisplatin, bleomycin and
adriamycin. In addition, known vascularization inhibitors include
indomethacin, medroxyprogesterone, a combination of cortisone and
heparin and crude extract of bovine cartilage or aortic wall.
However the occurrence of fungi or cells resistant to conventional
antibiotics or antitumor agents has made it necessary to
continuously develop novel ones. On the other hand, no
vascularization inhibitor so far has been put into practical use as
a medicine. Therefore it has been required to develop a novel
substance having antifungal, antitumor and
vascularization-inhibitory effects.
SUMMARY OF THE INVENTION
Under these circumstances, we have examined various metabolites of
microorganisms and consequently found that a strain belonging to
the genus Streptomyces produces a novel antibiotic NK86-0279 of the
formula (I): ##STR2## which exhibits antifungal, antitumor,
vascularization-inhibitory and insecticidal activity.
The present invention has been completed based on the above
finding.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows ultraviolet absorption spectra of NK86-0279
wherein------represents a 20 .mu.g/ml solution thereof in methanol;
-----represents a 20 .mu.g/ml solution thereof in 0.1 N
hydrochloric acid/90% methanol; and------represents a 20 .mu.g/ml
solution thereof in 0.1 N caustic soda/90% methanol.
FIG. 2 shows an infrared absorption spectrum of NK86-0279
determined in the form of a potassium bromide tablet.
FIG. 3 shows a hydrogen nuclear magnetic resonance spectrum of
NK86-0279 determined in deuterated chloroform.
FIG. 4 is a carbon nuclear magnetic resonance spectrum of NK86-0279
determined in deuterated chloroform.
DETAILED DESCRIPTION OF THE INVENTION
The novel antibiotic NK86-0279 as described above can be obtained
by culturing an NK86-0279 producing strain belonging to the genus
Streptomyces to thereby accumulate NK86-0279 and then recovering
the same from the culture medium. A typical example of the
NK86-0279 producing strain is Streptomyces bottropensis NK86-0279
isolated from soil in Funabashi, Chiba prefecture in August 1986
(FERM BP-1785), deposited at the Fermentation Research Institute
Agency of Industrial Science and Technology, 1-3, Higashi 1-chrome
Tsukuba-shi Ibaraki-Ken, 305, Japan which will be simply called
NK86-0279 strain hereinafter.
The NK86-0279 strain has the following mycological properties.
Mycological properties of NK86-0279 strain:
1. Morphology
When observed under a microscope, this strain shows spiral hyphae
from branched aerial hyphae and no whirl. A matured spore chain
comprises 20 or more spores (0.6-0.8.mu..times.1.2-1.4 .mu.) of a
smooth surface. No sporangium is observed.
2. Growth in various media
Each color is expressed according to the standard determined by
Nippon Shikisai Kenkyusho.
(1) Sucrose/nitrate agar medium (cultured at 27.degree. C.)
White aerial hyphae are formed on pale yellowish brown vegetative
mycelium. A slightly yellowish soluble pigment is observed.
(2) Glucose/asparagine agar medium (cultured at 27.degree. C.)
White to light brownish gray aerial hyphae are formed on pale
yellow to pale yellowish brown vegetative mycelium. No soluble
pigment is observed.
(3) Starch/inorganic salt agar medium (ISP-medium 4, cultured at
27.degree. C.)
Light brownish gray to brownish gray aerial hyphae are formed on
pale yellowish brown vegetative mycelium. A slightly brown soluble
pigment is observed.
(4) Tyrosine agar medium (ISP-medium 7, cultured at 27.degree.
C.)
Grayish white to light brownish gray aerial hyphae are formed on
dark brownish gray vegetative mycelium. A dark brown soluble
pigment is observed.
(5) Nutrient agar medium (cultured at 27.degree. C.)
No aerial hypha is formed on pale yellowish brown vegetative
mycelium. A slightly brown soluble pigment is observed.
(6) Yeast/malt agar medium (ISP-medium 2, cultured at 27.degree.
C.)
Grayish white to brownish gray aerial hyphae are formed on pale
yellowish brown vegetative mycelium. A slightly brown soluble
pigment is observed.
(7) Oatmeal agar medium (ISP-medium 3, cultured at 27.degree.
C.)
Grayish white to light brownish gray aerial hyphae are formed on
colorless vegetative mycelium No soluble pigment is observed.
(8) Starch agar medium (cultured at 27.degree. C.)
White to light brownish gray aerial hyphae are formed on pale
yellow to pale yellowish brown vegetative mycelium. A lightly brown
soluble pigment is observed.
(9) Calcium malate agar medium (cultured at 27.degree. C.)
White aerial hyphae are slightly formed on pale yellow to pale
yellowish brown vegetative mycelium. No soluble pigment is
observed.
(10) Glycerol/asparagine agar medium (ISP-medium 5, cultured at
27.degree. C.)
Brownish white to light brownish gray aerial hyphae are formed on
pale yellow vegetative mycelium. No soluble pigment is
observed.
(11) Glycerol/nitrate agar medium (cultured at 27.degree. C.)
White aerial hyphae are formed on pale yellowish brown vegetative
mycelium. A slightly brown soluble pigment is observed.
(12) Gelatin stab culture
No aerial hypha is formed on pale yellow to pale yellowish brown
vegetative mycelium cultured either in a gelatin medium at
20.degree. C. or in a glucose/peptone gelatin medium at 24.degree.
C. A brown soluble pigment is observed.
(13) Skimmed milk (cultured at 32.degree. C.)
White aerial hyphae are formed on pale yellow to pale yellowish
brown vegetative mycelium. A brown soluble pigment is observed.
3. Physiological properties
(1) Growth temperature range
The test strain is cultured in a yeast/starch agar medium
comprising 1.0% of soluble starch, 0.2% of yeast extract (mfd. by
NIPPON SEIYAKU CO., LTD.) and 2.0% of agar powder (mfd. by EIKEN
CHEMICAL CO., LTD.) (pH 7.0) at 5, 10, 24, 27, 32, 37 and
45.degree. C. As a result, it grows at all of these temperatures
except 5 and 45.degree. C. The optimum growth temperature seems to
range from 24.degree. to 32.degree. C.
(2) Liquefaction of gelatin
The test strain is cultured in a 15% gelatin medium at 20.degree.
C. and in a glucose/peptone/gelatin medium at 27.degree. C. As a
result, the liquefaction of the gelatin is observed from the 17th
day in each case. Thus the liquefying effect of the test strain on
gelatin is relatively low.
(3) Hydrolysis of starch
The test strain is cultured in a starch/inorganic salt agar medium
at 27.degree. C. and in a starch agar medium at 27.degree. C. As a
result, the hydrolysis of the starch is observed from the 10th day
in each case. Thus the hydrolyzing action of the test strain on
starch is moderate.
(4) Coagulation/peptonization of skimmed milk
The test strain is cultured in defatted milk at 32.degree. C. As a
result neither coagulation nor peptonization of the skimmed milk is
observed even on the 21st day.
(5) Formation of melanin-like pigment
The test strain is cultured in a trypton/yeast/broth ISP-medium 1
at 27.degree. C.; in a peptome/yeast/iron agar medium, ISP-medium 6
at 27.degree. C.; and in tyrosine agar medium, ISP-medium 7 at
27.degree. C. As a result, a melanin-like pigment is formed in each
case.
(6) Utilization of carbon source
The test strain is cultured in a Pridham-Gottlieb agar medium
ISP-medium 6 at 27.degree. C. As a result, it is found that this
strain utilizes glucose, L-arabinose, D-xylose, sucrose, inositol,
D-fructose, D-mannitol, rhamnose, raffinose and galactose.
(7) Dissolution of calcium malate
The test strain is cultured in a calcium malate agar medium at
27.degree. C. As a result, it exhibits a dissolution effect
thereon.
(8) Reduction of nitrate
The test strain is cultured in aqueous peptone containing 0.1% of
potassium nitrate ISP-medium 8 at 27.degree. C. The result is
negative.
These properties indicate that the NK86-0279 strain belongs to the
genus Streptomyces and contains LL-2;6-diaminopimelic acid in the
cell wall. It shows spiral aerial hyphae without any sporangium nor
whirl. The surface of a spore is smooth. It forms grayish white to
light brownish gray aerial hyphae on pale yellow to pale yellowish
brown vegetative mycelium in various media. A slightly brown
soluble pigment is observed. It forms a melanin-like pigment and
exerts a relatively low effect of decomposing protein and a
moderate effect of hydrolyzing starch.
A known strain which closely relates to the NK86-0279 strain based
on the abovementioned properties is Streptomyces bottropensis (cf.
International J. Systematic Bacteriol., 19, 410 (1969)). These two
strains are completely coincident with each other in, for example,
the colors of aerial hyphae on various media, utilization of sugars
and formation of a melanin-like pigment. These facts indicate that
the NK86-0279 strain belongs to Streptomyces bottropensis. Thus it
is named Streptomyces bottropensis NK86-0279.
The NK86-0279 productivity of the Streptomyces bottropensis
NK86-0279 to be used in the present invention can be enhanced
through various mutagenic techniques commonly employed in the art,
such as irradiation with UV light or .sup.60 Co, the use of a
mutagenic agent selected from among, for example, nitrogen mustard,
nitrous acid, N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and
2-aminopterin, transduction or cell fusion.
The antibiotic NK86-0279 of the present invention may be produced
by culturing a microorganism, which belongs to the genus
Streptomyces and is capable of producing the antibiotic NK86-0279,
in a medium to thereby accumulate said antibiotic in the medium and
then recovering the same.
Although the culture may be carried out, as a rule, in the same
manner as the one employed in the culture of actinomycetes,
submerged culture in a liquid medium is generally advantageous. Any
medium may be used therefor so long as it contains nutrient sources
to be utilized by the NK86-0279 strain.
As the nutrient sources, those conventionally employed in culturing
actinomycetes may be employed. Examples thereof include carbon
sources such as glucose, galactose, mannitol, dextrin, starch,
starch syrup (hydrolysis of starch by malt), soybean oil and
mixtures thereof; and inorganic and organic nitrogen sources such
as ammonium chloride, ammonium sulfate, urea, ammonium nitrate,
sodium nitrate, peptone, meat extract, yeast extract, dry yeast,
corn steep liquor soybean oil cake, oatmeal, casamino acids, bacto
soytone, soluble vegetable protein and mixtures thereof. The medium
may further contain inorganic salt(s) such as common salt,
magnesium sulfate, copper sulfate, zinc sulfate, manganese
chloride, calcium carbonate and phosphates, if required.
Furthermore it may contain organic metaerial(s) capable of
promoting the production of NK86-0279, such as nucleic acids, amino
acids and vitamins, as well as inorganic material(s). When
considerable foaming is observed during the culture, a vegetable
oil such as soybean oil or linseed oil or a petroleum defoaming
agent such as Pronal 1 (mfd. by Toho Chemical Industry Co., Ltd.)
or Silicone KM-70 (mfd. by The shin-Etsu Chemical Co., Ltd.) may be
optionally added to the culture medium. It is preferable to effect
the culture at a temperature of 25.degree. to 30.degree. C. under
neutral or slightly acidic conditions. The aimed NK86-0279 may be
produced and accumulated in a liquid medium after continuing the
culture for three to six days in general. When the amount of the
product reaches the maximum, the culture is ceased and the mycelia
are filtered. From the mycelia thus obtained, the aimed product is
isolated and purified.
The isolation and purification of the antibiotic from the mycelia
may be carried out in a manner commonly employed for the isolation
and purification of a microbial metabolite from mycelia producing
the same. Although the NK86-0279 is soluble in organic solvents
such as methanol, acetone, ethyl acetate and ethanol, it is hardly
soluble in water. Thus it may be purified by a method employed for
purifying fat-soluble materials.
Namely, the isolation and purification of the aimed NK86-0279 may
be carried out by appropriately combining, for example, extraction
with various organic solvents and chromatography with the use of
silica gel or Sephadex.sup..RTM. LH-20.
For example, the culture medium is filtered to thereby collect the
mycelia, which are then extracted with acetone twice The acetone
solution is concentrated to dryness in vacuo and the residue is
washed with n-hexane.
The brown crude powder thus obtained is dissolved in chloroform and
purified by silica gel column chromatography. A chloroform/methanol
mixture (50:1) is employed as a developing solvent therefor.
Then active fractions are combined and dried to thereby give a
brown powder. This powder is dissolved in a mixture of
hexane/acetone (3:2) and purified by silica gel column
chromatography again.
A hexane/acetone mixture (3:2) is employed as a developing solvent
therefor. The active fractions are combined and concentrated to
dryness to thereby give a pale yellow powder.
The obtained powder is dissolved in methanol and chromatogrpahed on
a Sepahdex.sup..RTM. LH-20 column which has been previously
equilibrated with said solvent. Active fractions are Combined and
concentrated to dryness to thereby give colorless powder. This
powder is dissolved in a mixture of methanol/water and crystallized
to give the aimed NK86-0279 in the form of a colorless crystal. The
NK86-0279 titers during the culture and in the crude fractions are
determined by measuring the growth-inhibitory effect thereof on
HeLa S.sub.3 cells by the dye exlusion test.
The NK86-0279 thus obtained has the following physicochemical
properties:
(1) appearance: colorless crystal,
(2) molecular weight: FD-MS m/z; 819 (M+H).sup.+,
(3) elemental analysis: carbon: 66.54%, hydrogen: 9.29% and oxygen:
24.06%,
(4) molecular formula: C.sub.46 H.sub.74 O.sub.12
(5) melting point: 157.0.degree.-159.0.degree. C.
(6) specific rotation: [.alpha.]D 20=-47.0 (C 1.00, methanol)
(7) solubility: soluble in methanol, acetone, ethyl acetate and
dimethyl sulfoxide while hardly soluble in water and hexane;
(8) Rf value in silica gel (Kiesel gel 60 F.sub.254, 0.25 mm, mfd.
by Merck) thin layer chromatography: 0.37 and 0.53 by using
developing solvent systems of hexane/acetone (3:2; v/v) and
chloroform/methanol (30:1; v/v), respectively,
(9) ultraviolet absorption spectra: as shown in FIG. 1, ##EQU1##
(10) infrared absorption spectrum (determined in the form of a
potassium bromide tablet): as shown in
FIG. 2, the absorption maxima (cm.sup.-1) are as follows:
______________________________________ 3525, 3500, 3455, 2955,
2930, 2870, 2340, 2160, 1920, 1840, 1730, 1700, 1650, 1640, 1450,
1410, 1395, 1375, 1365, 1345, 1300, 1290, 1270, 1230, 1200, 1180,
1170, 1125, 1115, 1110, 1100, 1090, 1070, 1050, 1010, 1005, 985,
975, 950, 920, 910, 900, 880, 865, 840, 820, 800, 785, 770, 745,
720, 705, 675. ______________________________________
(11) hydrogen unclear magnetic resonance spectrum determined in
deuterated chloroform: as shown in FIG. 3,
(12) carbon nuclear magnetic resonance spectrum determined in
deuterated chloroform: as shown in FIG. 4, the chemical shift
(.delta.) are as follows:
______________________________________ 220.26, 220.14, 203.12,
164.98, 149.11, 137.20, 132.24, 130.63, 129.87, 122.58, 101.13,
83.10, 76.01, 72.99, 72.67, 72.04, 71.02, 67.10, 64.60, 46.60,
46.14, 45.96, 43.95, 41.87, 41.74, 40.34, 38.38, 36.79 (x2), 35.99,
33.63, 31.20, 30.70, 28.68, 25.04, 21.88, 21.06, 17.91, 14.55,
13.98, 13.11, 13.08, 12.11, 9.44, 8.40, 5.99, and
______________________________________
(13) color reactions: positive in molybdatophosphoric acid,
sulfuric acid and potassium permanganate reaction while negative in
Pauly and Rydon-Smith reactions.
As will be described hereinafter, the compound of the present
invention is expected to be useful as a medicine such as antifungal
agent, antitumor agent or vascularization inhibitor and as
aninsecticide. When the compound of the present invention is to be
used for a medicine, it may be either employed alone or mixed with
excipient(s) and formulated into, for example, injection, oral drug
or suppository. Any pharmaceutically acceptable excipient may be
used therefor and the type and composition thereof may be
appropriately determined depending on the pathway or method of
administration. For example, liquid excipients include water,
alchols and vegetable, animal and synthetic oils such as soybean
oil, peanut oil, sesami oil and mineral oils, while solid
excipients include sugars such as maltose and sucrose, various
amino acids, cellulose derivatives such as hydroxypropylcellulose
and organic acid salts such as magnesium stearate.
In order to formulate the antibiotic of the present invention into
an injection, it is preferable to use excipient(s) such as
phyisiological saline solution, various buffers, solutions of
sugars, for example, glucose, inositol and mannitol and glycols
such as ethyleneglycol and polyethylene glycol. It is also possible
to lyophilize the antibiotic of the present invention together with
excipient(s) such as sugars, for example, inositol, mannitol,
glucose, mannose, maltose and sucrose and amino acids such as
phenylalanine. The lyophilized preparation may be dissolved in a
solvent suitable for injection, for example, sterilized water,
glucose solution, electrolyte solution or amino acid solution and
then intravenously or intramuscularly administered.
In order to prepare an oral drug, the antibiotic of the present
invention is formulated into various forms such as tablets,
capsules, powder, granules, solution or dry syrup together with the
abovementioned liquid or solid excipient(s). It is further possible
to formulate the same into pellets for endermatic or mucosal
administration.
The compound of the present invention may be incorporated into a
preparation generally in an amount of 0.001 to 1% by weight,
preferably 0.01 to 0.1% by weight. For example, an injection may
contain 0.01 to 0.05% by weight of the same, while an oral drug may
contain 0.005 to 1% by weight, preferably 0.05 to 0.5% by weight,
of the same and the balance of excipient(s).
The dose of the antibiotic of the present invention should be
determined depending on the age, body weight and condition of a
patient as well as the purpose of the treatment. Generally
speaking, it may by administered parenterally at a dose of 0.1 to 5
ug/kg per day, or orally at a dose of 0.5 to 50 ug/kg per day. The
acute toxicity (LD.sub.50) of NK86-0279 on mice is 1.67 mg/kg
(i.p.).
When the compound of the present invention is to be used for
agrochemicals such as an insecticide or fungicide, it may be
employed alone if desired, but they are generally formulated by
blending suitable adjuvants to improve or stabilize the effects
thereof and used as such or after being diluted if necessary. The
compounds of the invention can be formulated in the conventional
manners well-known in the art in any convenient form such as dust,
granule, micro granule, wettable powder, flowable, emulsion,
microcapsule, oil, aerosol, heating fumigant (e.g. mosquito
repellent of an incense type or electric type), fuming agent such
as fogging, non-heating fumigant, or toxic feed.
Examples of said adjuvants are carrier (i.e. diluent) and other
adjuvants such as a spreader, emulsifying agent, wetting agent,
dispersing agent, fixing agent or disintegrator.
The content of active ingredients in the formulation of the present
invention varies depending on the conditions of use such as
formulation form or application method, and is usually from 0.2 to
95% by weight, preferably from 0.5 to 80% by weight, although the
active ingredient may be used alone in a special case.
The composition of the present invention may be used in an amount
which depends on the conditions such as formulation form, season or
method for application. Generally, it is used in an amount of 10 to
300 g/10 a (a=100 m.sup.2), and preferably 15 to 200 g/100 a (in
terms of the active ingredient) for the control of insect pests in
ornamental forest or livestock and in an amount of 2 to 200
mg/m.sup.2, preferably 5 to 100 mg/m.sup.2 (in terms of the active
ingredient) for the purpose of exterminating hygienic insect pests.
For example, from 15 to 120 g/10 a of the active ingredient is used
in the case of dust, 30 to 240 g/10 a thereof is used in the case
of granule and 40 to 250 g/10 a thereof is used in the case of
emulsion or wettable powder. However, it may be possible, or even
necessary, to use the active ingredient in an amount which is
outside the range as specified above, in a special case.
Now the effects of the compound of the present invention will be
described.
TEST EXAMPLE 1
The antifungal spectrum of the compound of the present invention
was examined.
Table 1 shows the antifungal spectrum of NK86-0279 on a Czapek Dox
solution agar.
As shown in Table 1, NK86-0279 exerted strong growth-inhibitory
effects on fungi including Penicillium chrysogenum, Aspergillus
oryzae and Torula herbarum.
TABLE 1 ______________________________________ Minimum inhibitory
Test strain concentration (ug/ml)
______________________________________ Mucor javanicus >100
Aspergillus niger 6.25 Aspergillus oryzae 3.13 Penicillium
chrysogenum 1.56 Torula herbarum 3.13 Botrytis cinerea(*) 12.5
Fusarium roseum f. sp. 6.25 Rhizopus hangchao 6.25 Glomerella
cingulata 6.25 Sporobolomyces salmonicolor 6.25 Saccharomyces
cerevisiae >100 Candida albicans >100
______________________________________ (*)potato.sucrose agar
medium was used.
TEST EXAMPLE 2
The growth-inhibitory effect of the compound of the present
invention on HeLa S.sub.3 culture cells was examined. A 96-well
plate was inoculated with HeLa S.sub.3 cells at a ratio of
1.5.times.10.sup.3 cells/well and incubated in an incubator at
37.degree. C. under 5% CO for 24 hours. One day after the
inoculation, the compound of the present invention was added to the
culture medium at various concentrations. Three days after the
addition, cells in each well were counted by the dye exclusion
method to thereby evaluate the growth-inhibitory effect of the
compound of the present invention at various concentrations on HeLa
S.sub.3 cells.
Table 2 shows the results. Thus the IC.sub.50 of the compound of
the present invention was 0.0027 .mu.g/ml and it exerted a strong
growth-inhibitory effect on the HeLa S.sub.3 cells.
TABLE 2 ______________________________________ Growth-inhibitory
effect of NK86-0279 on HeLa S cells at various concentrations
Concentration of the compound Growth-inhibitory ratio (.mu.g/ml)
(%) ______________________________________ 0.00064 22.9 0.0032 53.5
0.016 61.2 0.08 73.5 0.4 81.2
______________________________________
TEST EXAMPLE 3
3-1
The growth-inhibitory effects of the compound of the present
invention on mouse colonic cancer cells (Colon 26) and human
colonic cancer cells (SW1116) were examined. A 96-well plate was
inoculated with mouse colonic cancer cells at a ratio of
1.5.times.10.sup.3 cells per well, while another 96-well plate was
inoculated with human colonic cancer cells at a ratio of
3.0.times.10.sup.3 per well. These cells were incubated in an
incubator at 37.degree. C. under 5% CO.sub.2 for 24 hours. Then the
compound of the present invention was added thereto at various
concentrations. Cells in each well were counted by the dye
exclusion method 65 hours and 96 hours after the addition to
thereby evaluate the growth-inhibitory effect of the test compound
on the mouse colonic cancer cells and that on the human colonic
cancer cells.
Table 3-1 shows the results. Thus the IC.sub.50 of the compound of
the present invention on the mouse colonic cancer cells was less
than 0.0061 .mu.g/ml, while that on the human colonic cancer cells
was 0.021 .mu.g/mlm showing a strong growth-inhibitory effect in
each case.
TABLE 3-1 ______________________________________ Conc of the
Growth-inhibitory ratio (%) compound mouse colonic cancer human
colonic cancer (.mu.g/ml) cells (Colon 26) cells (SW1116)
______________________________________ 100 103.7 90.7 25 108.4 91.9
6.25 104.9 80.8 1.56 94.2 52.2 0.39 87.6 53.5 0.098 86.1 54.6 0.024
87.5 51.7 0.0061 84.6 33.3
______________________________________
3-2
The anti-cancer action of the compound of the present invention
against the cancer of the mouse colon (colon 26) was examined. A
cancer debris of mouse colon having an area of 2 mm.sup.3 was
grafted into a subcutaneous portion of the left body side of a 6
week-old male BALB/c mouse, and the compound of the present
invention was intraperitoneally administered to the mouse in
various concentrations every day from the next day of the graft
once a day for 9 days. The general conditions of the mouse were
observed every day after the initiation of the administration. The
percentage prolongation of life (T/C) of the mouse with respect to
the compound of the present invention was determined from the ratio
of the average survival time on the groups of administration of the
compound of the present invention relative to that on the control
group.
The results are shown in Table 3-2. The compound of the present
invention exhibited a maximum percentage prolongation of life of
153% in a group to which the compound of the present invention had
been administered in an amount of 0.25 mg/kg, i.e., exhibited a
significant antitumor effect as compared with the control
group.
TABLE 3-2 ______________________________________ Dose of the
compound T/C (mg/kg) (%) ______________________________________
0.000 100 0.063 136 0.125 130 0.250 153 0.500 143
______________________________________
TEST EXAMPLE 4
4-1
The growth-inhibitory effects of the compound of the present
invention on mouse pulmonary cancer cells (LL) and human plumonary
cancer cells (PC-3) were examined.
A 96-well plate was inoculated with mouse pulmonary cancer cells at
a ratio of 8.0.times.10.sup.2 cells per well, while another 96-well
plate was inoculated with human pulmonary cancer cells at a ratio
of 2.5.times.10.sup.3 per well. These cells were incubated in an
incubator at 37.degree. C. under 5% CO.sub.2 for 24 hours. Then the
compound of the present invention was added thereto at various
concentrations. Cells in each well were counted by the dye
exclusion method three days after the addition to thereby evaluate
the growth-inhibitory effect of the test compound on the mouse
pulmonary cancer cells (LL) and that on the human pulmonary cancer
cells (PC-3).
Table 4-1 shows the results. Thus the IC.sub.50 of the compound of
the present invention on the mouse pulmonary cancer cells was
0.0098 .mu.g/ml, while that on the human pulmonary cancer cells was
2.13 .mu.g/ml, showing a strong growth-inhibitory effect in each
case.
TABLE 4-1 ______________________________________ Growth-inhibitory
ratio (%) mouse pulmonary human pulmonary Conc. of the cancer cells
cancer cells compound (.mu.g/ml) (LL) (PC-3)
______________________________________ 100 108.6 97.2 25 110.5 99.3
6.25 113.6 85.9 1.56 92.1 38.1 0.098 50.0 28.0 0.024 30.4 25.1
0.061 -5.7 12.0 ______________________________________
4-2
The anti-cancer action of the compound of the present invention
against mouse lung cancer (lewis lung carcinoma) and mouse breast
cancer (Ehrlich B) was examined. 1.times.10.sup.6 mouse lung cancer
cells or mouse breast cancer cells were grafted into a subcutaneous
portion of the groin of an eight week-old male BDF.sub.1 or ICR
mouse, and the compound of the present invention was
intraperitoneally administered to the mouse in various
concentrations every day from two days after the graft once a day
for 9 days. On the second day after completion of the
administration, the minor axis and the major axis of the tumor were
measured, and the volume of the tumor was determined by using the
following equation: (minor axis).sup.2 .times.(major axis).sup.2
/2. Then, the percentage inhibition of proliferation of each tumor
with respect to the compound of the present invention was
determined from the ratio of the volume of the tumor on the groups
of administration of the compound of the present invention relative
to that on the control group.
The results are shown in Table 4-2. The compound of the present
invention exhibited a maximum percentage inhibition of
proliferation of 59.6% in the mouse lung cancer group to which the
compound of the present invention had been administered in an
amount of 0.5 mg/kg and 67% in the mouse breast cancer group to
which the compound of the present invention had been administered
in an amount of 0.25 mg/kg, i.e., exhibited an excellent antitumor
effect against each tumor.
TABLE 4-2 ______________________________________ Dose of
Growth-inhibitory ratio (%) this mouse mouse compound pulmonary
breast (mg/kg) cancer cancer ______________________________________
0.500 59.6 61.7 0.250 8.9 67.0 0.125 57.1 44.3 0.063 46.3 -0.4
0.032 -- 22.5 0.000 0.0 0.0
______________________________________
TEST EXAMPLE 5
5-1
The growth-inhibitory effects of the compound of the present
invention on mouse melanoma cells (B16) and human melanoma cells
(A375) were examined. A 96-well plate was inoculated with mouse
melanoma cells at a ratio of 1.5.times.10.sup.3 cells per well,
while another 96-well plate was inoculated with human melanoma
cells at a ratio of 1.0.times.10.sup.3 per wall. These cells were
incubated in an incubator at 37.degree. C. under 5% CO.sub.2. Then
the compound of the present invention was added thereto at various
concentrations. Cells in each well were counted by the dye
exclusion method 65 hours after the addition to thereby evaluate
the growth-inhibitory effect of the test compound on the mouse
melanoma cells and that on the human melanoma cells.
Table 5-1 shows the results. Thus the IC.sub.50 of the compound of
the present invention on the mouse melanoma cells was 1.62
.mu.g/ml, while that on the human melanoma cells was 1.87 .mu.g/ml,
showing a growth-inhibitory effect in each case.
TABLE 5-1 ______________________________________ Conc. of the
compound Growth-inhibitory ratio (%) (.mu.g/ml) mouse melanoma
(B16) human melanoma (A375) ______________________________________
100 105.2 104.7 25 105.2 104.9 6.25 89.7 83.6 1.56 48.9 44.4 0.39
45.3 43.7 0.098 43.1 49.4 0.024 36.8 39.7 0.0061 5.3 19.7
______________________________________
5-2: in-vivo test by intraperitoneally administration.
The anti-cancer action of the compound of the present invention
against mouse malignant melanoma (B16 melanotic melanoma) was
examined 4.times.10.sup.5 mouse malignant melanoma cells were
inoculated into a subcutaneous portion of the right body side of a
8 week-old male BDF.sub.1 mouse, and the compound of the present
invention was intraperitoneally administered to the mouse in
various concentrations every day from the next day of the
inoculation once a day for 10 days. On the 11 days after the
inoculation the minor axis and the major axis of the tumor were
measured, and the volume of the tumor was determined by using the
following equation: (minor axis).sup.2 .times.(major axis).sup.2 /2
Then, the percentage inhibition of proliferation of each tumor with
respect to the compound of the present invention was determined
from the ratio of the volume of the tumor on the groups of
administration of the compound of the present invention relative to
that on the control group.
The results are shown in Table 5-2. The compound of the present
invention exhibited a maximum percentage inhibition of
proliferation of 80% in a group to which the compound of the
present invention had been administered in an amount of 1.0 mg/kg,
i.e., exhibited an excellent antitumor effect against the mouse
malignant melanoma.
TABLE 5-2 ______________________________________ Dose of Growth-
this inhibitory compound ratio (mg/kg) (%)
______________________________________ 1.000 80.0 0.577 60.4 0.333
53.2 0.192 54.7 0.111 38.4 0.064 40.5 0.037 41.4 0.021 19.3 0.012
50.7 0.000 0.0 ______________________________________
5-3: in-vivo test by intravenous administration
The anti-cancer action of the compound of the present invention
against mouse malignant melanoma (B16 melanotic melanoma) was
examined. 5.times.10.sup.5 mouse malignant melanoma cells were
inoculated into a subcutaneous portion of the right body side of a
6 week-old male C57BL/6 mouse, and the compound of the present
invention was intravenously administered to the mouse in various
concentrations every day from the next day or from 7 days after the
inoculation once a day for 5 days. On the 11 days after the
inoculation, the minor axis and the major axis of the tumor were
measured, and the volume of the tumor was determined by using the
following equation: (minor axis).sup.2 .times.(major axis).sup.2 /2
Then, the percentage inhibition of proliferation of each tumor with
respect to the compound of the present invention was determined
from the ratio of the volume of the tumor on the groups of
administration of the compound of the present invention relative to
that on the control group.
The results are shown in Table 5-3. The compound of the present
invention exhibited a maximum percentage inhibition of
proliferation of 81.9% in a group to which the compound of the
present invention had been administered in an amount of 0.4 mg/kg
from 7 days after the inoculation, i.e., exhibited an excellent
antitumor effect against the mouse malignant melanoma.
TABLE 5-3 ______________________________________ Growth-inhibitory
ratio (%) administered administered Dose of this from the next from
7 days compound day of the after the (mg/kg) inoculation
inoculation ______________________________________ 0.40 50.5 81.9
0.20 56.9 76.6 0.10 62.9 69.1 0.05 57.0 57.4 0.00 0.0 0.0
______________________________________
5-4: in-vivo test by peroral administration
The anti-cancer action of the compound of the present invention
against mouse malignant melanoma (B16 melanotic melanoma) was
examined. 5.times.10.sup.5 mouse malignant melanoma cells were
inoculated into a subcutaneous portion of the right body side of a
6 week-old male C57BL/6 mouse, and the compound of the present
invention was perorally administered to the mouse in various
concentrations every day from the next day or from 7 days after the
inoculation once a day for 5 days. On the 11 days after the
inoculation, the minor axis and the major axis of the tumor were
measured, and the volume of the tumor was determined by using the
following equation: (minor axis).sup.2 .times.(major axis).sup.2
/2. Then, the percentage inhibition of proliferation of each tumor
with respect to the compound of the present invention was
determined from the ratio of the volume of the tumor on the groups
of administration of the compound of the present invention relative
to that on the control group.
The results are shown in Table 5-4. The compound of the present
invention exhibited a maximum percentage inhibition of
proliferation of 94.6% in a group to which the compound of the
present invention had been administered in an amount of 1.0 mg/kg
from the next day of the inoculation, i.e., exhibited an excellent
antitumor effect against the mouse malignant melanoma.
TABLE 5-4 ______________________________________ Growth-inhibitory
ratio (%) administered administered Dose of this from the next from
7 days compound day of the after the (mg/kg) inoculation
inoculation ______________________________________ 8.0 66.0 67.0
4.0 64.5 75.5 2.0 69.5 57.4 1.0 94.6 72.3 0.5 90.5 44.7 0.0 0.0 0.0
______________________________________
TEST EXAMPLE 6
The growth-inhibitory effect of the compound of the present
invention on endothelial cells was examined. A 96-well plate was
inoculated with bovine adrenal capillary endothelial cells at a
ratio of 1.times.10.sup.4 cells per well. One day after the
inoculation, the compound of the present invention was added
thereto at various concentrations. Cells in each well were counted
with a Coulter counter three days after the addition to thereby
evaluate the growth-inhibitory effect of the test compound on the
capillary endothelial cells. Table 6 shows the results Thus the
IC.sub.50 of the compound of the present invention on the
endothelial cells was 0.0003 .mu.g/ml, showing a strong
growth-inhibitory effect.
TABLE 6 ______________________________________ Growth-inhibitory
ratio of NK86-0279 on capillary endothelial cells at various
concentrations Concentration of the compound Growth-inhibitory
ratio (.mu.g/ml) (%) ______________________________________
0.000128 35.4 0.00064 63.2 0.0032 78.4 0.016 89.7
______________________________________
TEST EXAMPLE 7
The vascularization-inhibitory effect of the compound of the
present invention was examined by corneal assay reported by M.A.
Gimbrone et al. (J. National Cancer Inst., 52, 413 (1974)). Namely,
the center of the cornea of the eye of a rabbit was incised with a
knife at a length of approximately 2 mm to thereby form a pouch.
Then a slow-releasing pellet containing 100 .mu.g of a erude
extract of Ehrlich ascites tumor cells, which had been
preliminarily prepared according to a method proposed by R. Langer
et al. (Nature, 263, 797 (1979)), was introduced into the pouch.
Further another slow-releasing pellet containing 0.3 to 81 .mu.g of
the compound of the present invention was introduced thereinto so
as to contact these pellets with each other. Then the degree of
vascularization was observed four, six, eight and ten days after
the introduction. As a result, the vascularization by the Ehrlich
ascites tumor cells crude extract was significantly retarded till
the fourth day in the groups to which 1 .mu.g or above of the
compound of the compound of the present invention had been
administered and till the eighth day in the groups to which 1.7
.mu.g or above thereof had been administered, compared with a
control group.
TEST EXAMPLE 8
The insecticidal action of the compound of the present invention
was examined by the following method.
(1) An imago of two-spoted spider mite acarids (a
Kelthane-resistant species) was used as a test vermin, and the
mortality rate thereof was assayed according to an ordinary method
(described on page 327 of "Noyaku Seibutsu Kenteiho" edited by
Toyoji Hosotsuji). Specifically, a solution of the compound of the
present invention in acetone (0.1%) was added to 0.5 ml of an
aqueous sucrose solution (1%) to have a predetermined
concentration. Ten two-spoted spider mite acarids were released in
a vessel (200-ml beaker), and the vessel was covered with a
paraffin film. The agent solution prepared above was put on the
paraffin film, and another paraffin film was put thereon. The
beaker was allowed to stand in a thermostatic chamber
(20.+-.1.degree. C.) for a predetermined period of time, and the
life and death of the test vermin was investigated.
(2) A larva of mosquito culices (a sensitive species) was used as a
test vermin, and the mortality rate thereof was assayed according
to an ordinary method (described on page 109 of "Noyaku Jikkenho I,
Sacchuzaihen" published by Soft Science, Inc.).
Specifically, 10 ml of well water was placed in a vessel (15-ml
Schale), and 5 larvae of mosquito culices (the third stage) were
released in the vessel. A solution of the compound of the present
invention in acetone (0.1%) was added thereto so as to have a
predetermined concentration. The vessel was allowed to stand in a
thermostatic chamber (26.+-.1.degree. C.) for a predetermined
period of time, and the life and death of the test vermin was
investigated. The results are shown in Table 8. As is apparent from
the Table, the compound of the present invention exhibited
acaricidal action and culicidal action.
TABLE 8 ______________________________________ Mortality rate (%)
Conc. of Imagos of the two-spotted Larvae of mosquito compound
spider mite after (ppm) after 2 days 2 days 6 days 9 days
______________________________________ 100 100 -- -- -- 10 100 100
100 100 1 100 0 0 0 0 0 0 0 0
______________________________________
The results suggest that the compound of the present invention
exerts antifungal, anticancer, vascularization-inhibitory and
insecticidal effects and thus may be highly useful as a novel
antifungal agent, antitumor agent vascularization inhibitor or
insecticide.
To illustrate a process for the preparation of the compound of the
present invention, the following Example will be given.
EXAMPLE 1
100 ml of a medium (pH 7.2) comprising 2% of soluble starch, 0.5%
of glucose, 0.5% of peptone, 0.5% of yeast extract, 0.05% of
potassium phosphate dibasic, 0.05% of magnesium sulfate and 0.5% of
soybean powder was pipetted into an Erlenmeyer flask (500 ml) and
sterilized in an autoclave at 120.degree. C. for 20 minutes. Then
it was inoculated with one platinum loopful of NK86-0279 strain
(FERM BP-1785). The strain was cultured under shaking at 190 rpm at
27.degree. C. in a rotary shaker for two days. Separately 100 ml of
a medium (pH 7.0) comprising 4% of glycerol, 0.5% of polypeptone,
0.3% of yeast extract powder, 0.5% of meat extract, 0.3% of sodium
chloride and 0.05% of magnesium sulfate was pipetted into a
Erlenmeyer flask (500 ml) and sterilized in an autoclave at
120.degree. C. for 20 minutes. Then 2 ml of the abovementioned
culture medium was inoculated into the latter flask and cultured
therein under shaking at 190 rpm at 27.degree. C. in a rotary
shaker for five days. After filtering the culture medium, 3 kg of
mycelia were obtained. These mycelia were washed with 3 L of
distilled water and 3 L of acetone was added thereto. After
stirring overnight, the mixture was extracted. The mycelia were
filtered and further extracted with 2 L of acetone. The acetone
extract thus obtained (4.8 L) was concentrated to dryness in vacuo
to thereby given 5.0 g of a crude extract. 4.2 g of this crude
extract was dissolved in chloroform and chromatographed on a silica
gel column (400 ml) which had been previously equilibrated with a
mixture of chloroform/methanol (50:1), by using said solvent.
Active fractions were combined and concentrated to dryness in vacuo
to thereby give 122.8 mg of a yellowish white crude powder.
Then this powder was dissolved in a mixture of hexane/acetone (3:2)
and chromatographed on a silica gel column (20 ml) which had been
previously equilibrated with said solvent. Active fractions were
combined and concentrated to dryness in vacuo to thereby give 68.5
mg of a pale yellowish white crude powder.
Then this crude powder was dissolved in methanol and
chromatographed on a Sephadex LH-20 column (150 ml) which had been
equilibrated with said solvent.
Active fractions were combined and concentrated to dryness in vacuo
to give a colorless powder. This powder was dissolved in a mixture
of methanol/water and crystallized to give 22.0 mg of the aimed
NK86-0279 in the form of a colorless crystal.
* * * * *